1. Field of the Invention
This invention pertains to the field of methods for obtaining specific and stable integration of nucleic acids into chromosomes of eukaryotes. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ΦC31 integrase.
2. Background
Genetic transformation of eukaryotes often suffers from significant shortcomings. For example, it is often difficult to reproducibly obtain integration of a transgene at a particular locus of interest. Homologous recombination generally occurs only at a very low frequency. To overcome this problem, site-specific recombination systems have been employed. These methods involve the use of site-specific recombination systems that can operate in higher eucaryotic cells.
Many bacteriophage and integrative plasmids encode site-specific recombination systems that enable the stable incorporation of their genome into those of their hosts. In these systems, the minimal requirements for the recombination reaction are a recombinase enzyme, or integrase, which catalyzes the recombination event, and two recombination sites (Sadowski (1986) J. Bacteriol. 165: 341-347; Sadowski (1993) FASEB J. 7: 760-767). For phage integration systems, these are referred to as attachment (att) sites, with an attP element from phage DNA and the attB element encoded by the bacterial genome. The two attachment sites can share as little sequence identity as a few base pairs. The recombinase protein binds to both att sites and catalyzes a conservative and reciprocal exchange of DNA strands that result in integration of the circular phage or plasmid DNA into host DNA. Additional phage or host factors, such as the DNA bending protein IHF, integration host factor, may be required for an efficient reaction (Friedman (1988) Cell 55:545-554; Finkel & Johnson (1992) Mol. Microbiol. 6: 3257-3265). The reverse excision reaction sometimes requires an additional phage factor, such as the xis gene product of phage λ (Weisberg & Landy (1983) “Site-specific recombination in phage lambda.” In Lambda II, eds. Hendrix et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp. 211-250; Landy (1989) Ann. Rev. Biochem. 58: 913-949.
The recombinases have been categorized into two groups, the λ integrase (Argos et al. (1986) EMBO J. 5: 433-44; Voziyanov et al. (1999) Nucl. Acids Res. 27: 930-941) and the resolvase/invertase (Hatfull & Grindley (1988) “Resolvases and DNA-invertases: a family of enzymes active in site-specific recombination” In Genetic Recombination, eds. Kucherlipati, R., & Smith, G. R. (Am. Soc. Microbiol., Washington D.C.), pp. 357-396) families. These vary in the structure of the integrase enzymes and the molecular details of their mode of catalysis (Stark et al. (1992) Trends Genetics 8: 432-439). The temperate Streptomyces phage ΦC31 encodes a 68 kD recombinase of the latter class. The efficacy of the ΦC31 integrase enzyme in recombining its cognate attachment sites was recently demonstrated in vitro and in vivo in recA mutant Escherichia coli (Thorpe & Smith (1998) Proc. Nat'l. Acad. Sci. USA 95: 5505-5510). The ΦC31 integration reaction is simple in that it does not require a host factor and appears irreversible, most likely because an additional phage protein is required for excision. The phage and bacterial att sites share only three base pairs of homology at the point of cross-over. This homology is flanked by inverted repeats, presumably binding sites for the integrase protein. The minimal known functional size for both attB and attP is ˜50 bp.
The Cre-lox system of bacteriophage P1, and the FLP-FRT system of Saccharomyces cerevisiae have been widely used for transgene and chromosome engineering in animals and plants (reviewed by Sauer (1994) Curr. Opin. Biotechnol. 5: 521-527; Ow (1996) Curr. Opin. Biotechnol. 7: 181-186). Other systems that operate in animal or plant cells include the following: 1) the R-RS system from Zygosaccharomyces rouxii (Onouchi et al. (1995) Mol. Gen. Genet. 247: 653-660), 2) the Gin-gix system from bacteriophage Mu (Maeser & Kahmann (1991) Mol. Gen. Genet. 230: 170-176) and, 3) the β recombinase-six system from bacterial plasmid pSM19035 (Diaz et al. (1999) J. Biol. Chem. 274: 6634-6640). By using the site-specific recombinases, one can obtain a greater frequency of integration.
However, these five systems suffer from a significant shortcoming. Each of these systems have in common the property that a single polypeptide recombinase catalyzes the recombination between two sites of identical or nearly identical sequences. The product-sites generated by recombination are themselves substrates for subsequent recombination. Consequently, recombination reactions are readily reversible. Since the kinetics of intramolecular interactions are favored over intermolecular interactions, these recombination systems are efficient for deleting rather than integrating DNA. Thus, a need exists for methods and systems for obtaining stable site-specific integration of transgenes. The present invention fulfills this and other needs.